Ethosuximide (ETX) is a common antiepileptic drug in the first line of absence epilepsy. In this study, for the first time, an economical and efficient electro‐membrane (EME) method for determination of ETX in a complex biological matrix using HPLC‐UV has been developed. Factors affecting conventional EME were evaluated. 1‐Octanol was immobilized in a polypropylene membrane and a voltage of 35 V was applied between two platinum electrodes for 15 min. The pH of acceptor and donor phases for ionization of ETX was adjusted to 13 and 11, respectively. Under optimal microextraction conditions, the enrichment factor was 21.02 and the linear range of ETX was 0.25 to 8.00 μg/mL with an acceptable R2 ≥ 0.9986. Inter‐day and intra‐day precision and accuracy of the suggested method were calculated with RSD < 9.5% and relative error <7.0%, respectively. The mean relative recovery of ETX in the human saliva and plasma samples was 81.68% and 74.47, respectively; while limit of detection and quantification concentrations were 0.08 and 0.25 μg/mL, respectively. Furthermore, to evaluate the application of the method, plasma and saliva samples of volunteers administering a single dose of ETX were analyzed successfully by EME‐HPLC‐UV method.